Larcombe, M. R. | Search bioRxiv

bioRxiv2022

Retrotransposon instability dominates the acquired mutation landscape of mouse induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) can in principle differentiate into any cell of the body, and have revolutionized biomedical research and regenerative medicine. Unlike their human counterparts, mouse iPSCs (miPSCs) are reported to silence transposable elements (TEs) and prevent TE-mediated mutagenesis. Here we applied short-read or Oxford Nanopore Technologies (ONT) long-read genome sequencing to 38 bulk miPSC lines reprogrammed from 10 parental cell types, and 18 single-cell miPSC clones. While single nucleotide variants and structural variants restricted to miPSCs were rare, we found 83 de novo TE insertions, including examples intronic to Brca1 and Dmd. LINE-1 (L1) retrotransposons were profoundly hypomethylated in miPSCs, beyond other TEs and the genome overall, and harbored alternative protein-coding gene promoters. Treatment with the L1 inhibitor lamivudine did not hinder reprogramming and efficiently blocked endogenous retrotransposition, as detected by ONT sequencing. These experiments reveal the complete spectrum and potential significance of mutations acquired by miPSCs.

genomics↗

bioRxiv2020

TINC - a method to dissect transcriptional complexes at single locus resolution - reveals novel Nanog regulators in mouse embryonic stem cells

Cellular identity is ultimately controlled by transcription factors (TFs), which bind to specific regulatory elements (REs) within the genome to regulate gene expression and cell fate changes. While recent advances in genome-wide epigenetic profiling techniques have significantly increased our understanding of which REs are utilized in which cell type, it remains largely unknown which TFs and cofactors interact with these REs to modulate gene expression. A major hurdle in dissecting the whole composition of a multi-protein complex formed at a specific RE is the shortage of appropriate techniques. We have developed a novel method termed TALE-mediated Isolation of Nuclear Chromatin (TINC). TINC utilizes epitope-tagged TALEs to isolate a specific genomic region from the mammalian genome and includes a nuclei isolation and chromatin enrichment step for increased specificity. Upon cross-linking of the cells and isolation of the chromatin, the target region is purified based on affinity purification of the TALE and associated nucleic acid and protein molecules can be subjected to further analyses. A key TF in the pluripotency network and therefore in embryonic stem cells (ESCs) is NANOG. It is currently not fully understood how Nanog expression is regulated and consequently it remains unclear how the ESC state is maintained. Using TINC we dissected the protein complex formed at the Nanog promoter in mouse ESCs and identified many known and numerous novel factors.

genomics↗